Project summary In Parkinson's Disease (PD), the most common neurodegenerative movement disorder that afflicts millions of people worldwide, the proteostasis network breaks down and fails to counter the misfolding of the small presynaptic protein ?-synuclein (?-syn). ?-Syn populates a range of misfolded structures ranging from soluble toxic oligomers to self-templating amyloid fibrils capable of initiating and propagating disease de novo. ?-Syn fibrils cluster into large cytoplasmic inclusions termed Lewy Bodies, a pathological hallmark of PD. Recently, we and others have discovered a series of human molecular chaperones, Hsp110, Hsp70, Hsp40, and HspB5 which can disaggregate ?-syn fibrils and reduce their toxicity. Whether this system can also disassemble toxic soluble ?-syn oligomers remains unclear. This endogenous disaggregase system likely becomes overwhelmed and fails to counter ?-syn misfolding in PD and related ?-synucleinopathies. Indeed, Hsp70 chaperones are often sequestered and depleted by excessive accumulation of misfolded proteins. Methods to stimulate the Hsp110, Hsp70, Hsp40, and HspB5 disaggregase machinery in the degenerating neurons of PD patients could reverse deleterious accumulation of ?-syn and provide a game-changing solution for PD. Thus, we propose that the Hsp110, Hsp70, Hsp40, and HspB5 disaggregase machinery represents a promising, novel PD- relevant target. We hypothesize that enhancing the activity of the Hsp110, Hsp70, Hsp40, and HspB5 disaggregase system with specific brain-penetrant small molecules will enable dissolution of toxic oligomeric and amyloid forms of ?-syn, and confer therapeutic benefits in PD. In the proposed studies, we will pursue two specific aims: (1) isolate brain-penetrant small molecules that enhance the ability of Hsp110, Hsp70, Hsp40, and HspB5 to disaggregate ?-syn oligomers and fibrils; and (2) Determine the ability of brain- penetrant, small-molecule enhancers of Hsp110, Hsp70, Hsp40, and HspB5 disaggregase activity to mitigate ?-syn misfolding and toxicity in primary neurons. This project makes an important first step toward exploring the feasibility of developing brain-penetrant small-molecule therapeutics that enhance the activity of the human ?-syn disaggregase machinery (Hsp110, Hsp70, Hsp40, and HspB5) as an alternative treatment strategy for PD. By the end of our studies, there will be a clear ?go/no go? decision for moving a brain-penetrant small- molecule enhancer of Hsp110, Hsp70, Hsp40, and HspB5 disaggregase activity into rodent models and ultimately PD patients. Small-molecule stimulation of the human protein-disaggregase machinery could reverse deleterious ?-syn misfolding in degenerating dopaminergic neurons and provide a transformative solution for PD and related ?-synucleinopathies including dementia with Lewy Bodies and multisystem atrophy. Importantly, brain-penetrant small-molecule enhancers of Hsp110, Hsp70, Hsp40, and HspB5 may also have important applications in other neurodegenerative disorders, including Alzheimer's disease.